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Ž .
Aquaculture 183 2000 161–177
www.elsevier.nlrlocateraqua-online
Lipid and fatty acid composition of early stages of
cephalopods: an approach to their lipid
requirements
Juan C. Navarro a,)
, Roger Villanueva b
a
( )
Instituto de Acuicultura de Torre de la Sal CSIC , E-12595 Ribera de Cabanes, Castellon, Spain
´
b
( )
Instituto de Ciencias del Mar CSIC , Paseo Juan de Borbon srn, E-08039 Barcelona, Spain
´
Accepted 10 August 1999
Abstract
We report here the main lipid classes and the fatty acid composition from the total lipids of
hatchlings of three cephalopod species: Sepia officinalis, Loligo Õulgaris and Octopus Õulgaris, as
well as the lipid composition of two selected crustaceans that have been used previously with
success as food resource for rearing cephalopod hatchlings: zoeae of Pagurus prideaux and the
mysidacean Acanthomysis longicornis. Additionally, we report the lipid class and fatty acid
composition of two cultures of O. Õulgaris paralarvae reared with enriched Artemia juveniles,
and with enriched Artemia juveniles plus a prepared pelleted diet. From their lipid composition
and that of their natural food, it can be deduced that cephalopod paralarvae and juveniles must
Ž .
require a food rich in polyunsaturated fatty acids PUFA , phospholipids and cholesterol and with
a moderate content in neutral lipids. There was a clear influence of the lipid composition of the
food on the lipids of cultured octopus paralarvae both at the level of lipid class and fatty acid
composition. The cultured octopus paralarvae showed a lower content of PUFA as compared with
the newly hatched individuals. Co-feeding techniques based on the use of polar lipid and PUFA
enriched Artemia together with palatable pellets seemed to be a possible way to improve
paralarval and juvenile cephalopod culture beyond the experimental scale. q 2000 Elsevier
Science B.V. All rights reserved.
Keywords: Lipids; Fatty acids; Cephalopods; Octopus; Squid; Cuttlefish
)
Corresponding author. Tel.: q34-964319500; fax: q34-964319509; e-mail: jcnavarro@iats.csic.es
0044-8486r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.
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PII: S0044-8486 99 00290-2
( )
J.C. NaÕarro, R. VillanueÕarAquaculture 183 2000 161–177
162
1. Introduction
Cephalopods have been cultured through the complete life cycle and multiple
generations only at an experimental scale or, in few numbers for biomedical research.
Ž
The cuttlefish Sepia officinalis Pascual, 1978; Boletzky, 1983; Forsythe et al., 1994;
. Ž .
Hanley et al., 1998 and Sepioteuthis lessoniana Lee et al., 1994 seem to be the most
easily cultured due, among other reasons, to the large size of the hatchlings. Many
cephalopod species, mostly squids and octopods, have a planktonic posthatching stage,
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and the term paralarva has been proposed for these stages Young and Harman, 1988 .
In other cephalopods, such as the cuttlefishes, hatchlings are large and immediately
benthic like the adults and thus are referred to as juveniles. Paralarvae and juveniles are
active swimmers and rapid growing forms that require substantial amounts of food
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Vecchione, 1991; Villanueva and Nozais, 1996; Villanueva et al., 1995 . In fact,
subadult and adult cephalopods show high metabolic rates and fast growth which are
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sustained by high feeding rates O’Dor and Wells, 1987 . Digestive capability of
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paralarval stages Boucaud-Camou and Roper, 1995, 1998 indicate a carnivorous diet
Ž . Ž .
as in juveniles Boucaud-Camou et al., 1985 and adults Boucher-Rodoni et al., 1987 .
The culture of cephalopods is becoming an important area of interest due to their
Ž y1 . Ž .
rapid growth )5% body weight day Forsythe and Van Heukelem, 1987 and high
market price. A suitable species is the common octopus Octopus Õulgaris, due to its
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rapid growth and high food conversion Mangold and Boletzky, 1973; Mangold, 1983 .
At present, the commercial culture of O. Õulgaris in Spanish waters is limited to
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ongrowing subadults captured from the wild, to adult sizes Iglesias et al., 1997;
.
Rama-Villar et al., 1997 . Octopus with planktonic stages like O. Õulgaris, have been
Ž
successfully reared through their paralarval stages only at an experimental scale Itami et
.
al., 1963; Villanueva, 1995 . The paralarval culture of octopus and early stages of most
cephalopod species, still being a bottleneck. The suitable food for rearing these early
stages has been an unresolved problem. At present, most of the live prey used for
successfully rearing early stages are collected from the sea, as zooplankton for paralarval
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squids Yang et al., 1986; Hanlon et al., 1979, 1989 or mysids and palaemonids for
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juvenile cuttlefishes and squids Forsythe et al., 1994; Lee, 1994 , but this method
requires a substantial input of time and effort. On the other hand, laboratory hatched
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decapod zoeae have been used as food for paralarval octopods and squids Itami et al.,
.
1963; Villanueva, 1994, 1995 , but the use of this food item requires a risky cumber-
some parallel culture of crustaceans that seems impractical beyond the experimental
scale systems.
Aside from the problems related to food size and quantity, there seem to be other
problems associated with food quality. Since cephalopods have a predominant amino
Ž .
acid metabolism Lee, 1994 , research on the lipid and fatty acid requirements have been
somewhat neglected to the point that these are scarcely known at present. Adult
Ž . Ž
cephalopods are rich in long chain polyunsaturated fatty acids PUFA Jangaard and
.
Ackman, 1965; Nash et al., 1978 , their oil, being a good source of these compounds
Ž
often used in aquaculture to supplement feeds Southgate and Lou, 1995; Knauer and
.
Southgate, 1997 . As far as we are aware, there are no reports on the fatty acid
composition of paralarval and juvenile cephalopods. According to the fatty acid compo-
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J.C. NaÕarro, R. VillanueÕarAquaculture 183 2000 161–177 163
sition of the adults, and to the generic composition of their natural food, the paralarvae
must require high amounts of these fatty acids. This hypothesis is further substantiated
by the fact that earlier attempts to culture octopus and squid paralarvae with the brine
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shrimp Artemia failed Boletzky and Hanlon, 1983; Hamazaki et al., 1991 . It is known
that Artemia, in general, lacks long chain PUFA essential for marine animals, like
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eicosapentaenoic acid EPA, 20:5ny3 which may be present in low amounts or absent
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in the lipid of the brine shrimp, and docosahexaenoic acid DHA, 22:6ny3 which is
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basically absent Navarro et al., 1992, 1993a .
The aim of the present work was to take a first insight on the nutritional requirements
of the paralarval and juvenile stages of cephalopods in culture. First, we determined the
main lipid classes and the fatty acid composition of the newly hatched individuals of
three cephalopod species that represent the three main cephalopod orders, all of them of
high commercial interest: the cuttlefish S. officinalis, the European squid L. Õulgaris
and the common octopus O. Õulgaris. Second, we analysed the same lipid composition
of two selected crustaceans that have been successfully used previously as food
resources for rearing cephalopod hatchlings, under experimental cultures: zoeae of the
hermit crab Pagurus prideaux and the mysidacean shrimp Acanthomysis longicornis
Ž .
Villanueva, 1994 . Finally, we report the lipid class and fatty acid composition of two
cultures of O. Õulgaris paralarvae reared with enriched Artemia juveniles, and with
enriched Artemia juveniles plus a prepared pelleted diet.
2. Materials and methods
2.1. Collection of material
Ž
Cephalopod eggs were collected off Vilanova i la Geltru, Barcelona NW Mediter-
´
.
ranean by means of the local ceramic pot fishery for octopus. The pot line was placed
near the coast, between 10 and 25 m depth. Egg masses of S. officinalis and egg
capsules of L. Õulgaris, attached to the fishery line were collected on board and
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transported to the lab the same day February 1998 . Eggs were incubated at ambient
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temperature mean: 15.28C, range: 14.7–15.68C , using an open-circuit of filtered sea
water at the ICM, Barcelona. During February and March 1998, active, healthy
individuals of the three cephalopod species: S. officinalis, L. Õulgaris and O. Õulgaris,
recently hatched in aquaria were collected for analysis.
Additionally, two crustacean species were analyzed for lipid and fatty acid composi-
tion. Ovigerous females of the hermit crab P. prideaux were collected from the by-catch
of the craft fishery from the same place and depth as above, in January 1998. Ovigerous
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females were transported to the lab, maintained as described in Villanueva 1994 and
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the recent hatched zoeae used for analysis. Mysids A. longicornis were collected in
May 1998, off Badalona, Barcelona, by a hand net, at 10 m depth using scuba diving.
The same day the individuals were preserved for analysis.
Freshly hatched individuals of cephalopods and crustaceans were collected and
preserved during the first 24 h after hatching in aquaria. The samples were collected
using a hand net, washed in freshwater, then placed in blotting paper to remove the
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J.C. NaÕarro, R. VillanueÕarAquaculture 183 2000 161–177
164
water, and weighed using an Ohaus Analytical Plus AP250D-O microbalance. Samples
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were preserved in chloroformrmethanol 2:1, vrv with 0.01% butylated hydroxy-
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toluene wrv as antioxidant. From each sample three aliquot replicates were used for
dry weight determinations after drying in an oven for 48 h at 1008C.
2.2. Rearing experiments of O. Õulgaris paralarÕae
O. Õulgaris broodstock collected from local craft fishery was maintained using the
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open-circuit seawater system of the ICM. Females 1–2 kg were placed with males of
similar size during 2–3 days to ensure mating. Fertilised females were isolated and
maintained in 200-l individual cylindrical plastic tanks. An overabundance of food was
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supplied using frozen crabs Carcinus maenas and sardine Sardina pilchardus . All
fertilised females were maintained in the dark with the aim of accelerating their sexual
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maturation Wells, 1978; Zuniga et al., 1995 . Some of them were also kept at warm
´˜
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temperature by heating the water to 19–218C with the same purpose Villanueva, 1995 .
Embryonic development of the egg masses and hatching time were controlled according
to the rearing needs by means of the temperature of the water, since this is the main
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factor that modulates the duration of the embryonic period Boletzky, 1989 .
A 700-l semi-closed seawater system connected to the open-circuit of the ICM was
used for the paralarval rearing experiments. Seawater was filtered through 0.2 mm and
daily renovation rates were 10–40%. The semi-closed system was equipped with a
biological filter, protein skimmer, UV lamps, ozonizer and temperature control. Two
rearing experiments of O. Õulgaris from hatching to 30 days were carried out. Artemia
biomass and pellets were used as food.
Ž .
Artemia AF, Artemia Systems nauplii were grown in seawater at 25–308C under
constant illumination during 7–10 days using 45-l cylindro-conical plastic tanks. Artemia
cultures were fed Dunaliella Õiridis algae to obtain Artemia biomass composed of 1–3
mm total length individuals. This prey size was selected because it had been previously
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used with success for rearing O. Õulgaris paralarvae Villanueva, 1994, 1995 . The
Artemia biomass was enriched in seawater for 24 h at 288C with one of the following
Ž . Ž . y1 Ž .
enrichment diets: a Diet SS: DC Super Selco Artemia Systems 0.6 g l and, b Diet
y1 Ž . y1
HB: DC Super Selco 0.6 g l , cod liver oil Acofarma Laboratories 0.6 g l , vitamin
Ž . y1 Ž . y1
C Sigma Products 0.3 g l , Hipramin-B Hipra Laboratories 0.4 ml l . Fifty
y1 y1 Ž .
thousand IU l of sodium G-penicillin and 50 mg l streptomycin sulphate Sigma
were added to both enrichment media to retard bacterial growth.
Ž .
The composition and formulation of the pelleted diet in dry weight % was: frozen
Ž . Ž . Ž .
euphausiids MBF 30%, squid Todarodes sagittatus powder Rieber and Son 18%,
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fish hydrolysate Divaq 18%, DC Super Selco 8%, phosphatidylcholine PC, Sigma
Ž . Ž . Ž
5%, cholesterol Sigma 3%, vitamin complex Kurios 5%, mineral complex Warner
. Ž . Ž .
Lambert 5%, gelatine Royal 7.975%, ethoxyquin Sigma 0.025%. Ingredients were
mixed with an electric blender, pelletized and dried at room temperature during 48 h.
The percentage of moisture was 6%. The pellets were sieved to obtain particle size of
250–500 mm and were stored at y208C before use. The colour of the pellet was reddish
brown.
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J.C. NaÕarro, R. VillanueÕarAquaculture 183 2000 161–177 165
Ž .
Fig. 1. Lipid class composition % of the total lipid of early stages of cephalopods: lpc: lysophosphatidyl-
choline, sm: shingomyelin, pc: phosphatidylcholine, ps: phosphatidylserine, pi: phosphatidylinositol, parcl:
phosphatidic acidrcardiolipin, pe: phosphatidylethanolamine, pigm: pigments, magrdag: monordi acylglyc-
erides, chol: cholesterol, tag: triacylglycerides, ffa: free fatty acids, se: sterol esters. Data are the mean of four
replicates. Error bars are the standard deviation.
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J.C. NaÕarro, R. VillanueÕarAquaculture 183 2000 161–177
166
Table 1
Ž . Ž .
Total lipid and total fatty acids % of dry weight , and fatty acids from total lipids wt.% of early stages of
cephalopods
Data are the means of three replicates. Standard deviations were below 10%.
0.0 are values below 0.09.
Fatty acid Hatchlings Paralarval culture
O. Õulgaris S. officinalis L. Õulgaris Octopus HB Octopus SS
-14:0 1.0 0.2 0.4 1.6 0.8
14:0 1.8 2.7 2.1 0.6 0.5
14:1 0.2 0.2
15:0 0.2 0.2 0.5 0.2 0.2
15:1 0.1
16:0 17.5 22.3 22.1 12.1 15.7
16:1ny9 0.7 0.1 0.3 0.2
16:1ny7 0.5 0.2 0.2 1.5 0.7
16:1ny5 0.4
16:2 0.1 0.2 0.2
17:0 1.4 1.0 1.7 1.4 1.2
16:3 0.1 0.1 0.2 0.3 0.3
16:4 0.1 0.7 0.1
18:0 6.3 8.7 9.1 11.4 12.2
18:1ny11 1.4 0.4 0.5 0.7
18:1ny9 4.0 4.0 2.8 11.7 5.3
18:1ny7 1.6 1.1 1.3 6.7 3.9
18:1ny5 0.2
18:2ny6 3.3 0.4 0.3 2.9 1.9
18:3ny6 0.1
18:3ny3 2.1 1.0
18:4ny3 0.1
20:0 0.1 0.2 0.3 0.3 0.2
20:1ny9 4.6 5.4 3.5 4.0 2.7
20:1ny7 0.4 0.3 0.3
20:2ny6 0.9 0.5 0.4 1.1 0.9
20:3ny6 0.2
20:4ny6 7.3 0.9 3.3 4.9 7.3
20:3ny3 0.7 0.4 0.6 1.2 1.2
20:4ny3 0.3 0.1
20:5ny3 12.6 14.3 14.5 17.4 14.4
22:0 0.1
22:1ny11 0.6 0.1 0.1 0.4 0.3
22:1ny9 0.5 0.4
22:2ny6 0.1 0.1
22:4ny6 0.9 0.2 0.2
22:5ny6 0.5 0.5 0.6 0.2 0.3
22:5ny3 1.7 0.5 1.1 1.5 1.0
22:6ny3 21.2 32.8 29.3 9.8 19.1
Total 90.9 96.6 96.2 95.0 94.3
Saturated 27.2 35.0 35.7 25.9 30.1
Monoenes 13.5 11.1 9.0 25.6 14.8
Polyunsaturated 49.2 50.3 51.1 41.9 48.6
ny3 36.2 48.0 45.5 32.5 37.0
ny6 12.9 2.1 4.7 8.9 11.1
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J.C. NaÕarro, R. VillanueÕarAquaculture 183 2000 161–177 167
Ž .
Table 1 continued
Fatty acid Hatchlings Paralarval culture
O. Õulgaris S. officinalis L. Õulgaris Octopus HB Octopus SS
PUFA ny3 36.2 48.0 45.5 30.2 35.9
PUFA ny6 9.6 1.7 4.5 6.0 9.1
ny3rny6 2.9 23.4 9.6 3.6 3.3
dharepa 1.7 2.3 2.0 0.6 1.3
% total fatty acids 4.6 4.6 4.1 7.2 5.4
% total lipid 13.4 12.5 10.8 25.1 13.7
2.2.1. O. Õulgaris paralarÕal culture HB
The rearing experiment started with a total of 1250 freshly hatched O. Õulgaris
paralarvae divided in two cylindric polyethylene plastic tanks. The characteristics of
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these tanks and general rearing conditions have been described elsewhere Villanueva,
. y1
1995 . Tank volume was 70 l and water flow 120 l h . Temperature ranged from 20 to
Ž . Ž .
228C. Artemia biomass Diet HB was supplied ad libitum 2–3 times a day from the
first day of culture to the end at 30 days.
2.2.2. O. Õulgaris paralarÕal culture SS
The experiment started with a total of 1600 freshly hatched O. Õulgaris paralarvae
divided in four cylindric PVC tanks. Tank volume was 25 l and water flow 80 l hy1
.
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Temperature ranged from 20.6 to 22.58C. As feeding regime Artemia biomass Diet SS
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was supplied ad libitum 2–3 times a day through the experimental period and pelleted
diet from day 10 to 30. Pellets were continuously supplied in excess 24 h dayy1
using
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an automatic delivery system Dohse Aquaristik .
In both experiments illumination was constant 24 h dayy1
. Tanks were cleaned daily
and dead animals removed and counted. At days 10, 20 and 30, samples of 15 paralarvae
Ž .
were cold-anaesthetised 2–48C and weighed after blotting on paper to remove the
water of the mantle cavity. Dry weights were obtained after drying the samples in an
oven for 48 h at 1008C.
2.3. Analytical
Ž .
Lipids were extracted using the method of Folch et al. 1957 . Lipid classes were
separated by high performance thin layer chromatography using the method of Olsen
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and Henderson 1989 , and quantified by densitometry Bio Rad GS 670 Imaging
. Ž
densitometer . For fatty acid analyses, aliquots were transmethylated overnight Christie,
. Ž .
1982 after the addition of 19:0 as an internal standard Sigma . Methyl esters were
Ž .
extracted with hexanerdiethyl ether 1:1, vrv , and purified by thin layer chromatogra-
Ž . Ž
phy Silica Gel G 60, Merck using hexanerdiethyl etherracetic acid 85:15:1.5,
.
vrvrv as solvent. The analyses of the methyl esters were carried out in a Fisons 8000
gas chromatograph equipped with a fused silica 30 m=0.25 mm open tubular column
Ž .
Tracer, TR-WAX, film thickness: 0.25 mm and a cold on-column injection system,
using helium as carrier gas, and a thermal gradient from 50 to 2208C. Peaks were
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J.C. NaÕarro, R. VillanueÕarAquaculture 183 2000 161–177
168
recorded in a personal computer linked to the analytical instrument using Chrom-Card
Ž .
software Fisons , quantified with the aid of the response factor of the internal standard
and identified by comparison with a well characterised marine oil.
3. Results
All the cephalopod paralarvae and juveniles were rich in polar lipid and cholesterol
Ž . Ž .
Chol. , the latter ranging from 18 to 25% of total lipid Fig. 1 . In general, PC,
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phosphatidylethanolamine PE and Chol. accounted for more than 60% of the total
Ž . Ž .
lipid. Squid and cuttlefish were richer in PC 34, 33% and PE 25, 22% as compared to
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newly hatched octopuses 21, 16% . The lipids of O. Õulgaris paralarvae, both newly
Ž .
hatched and cultured were richer in phosphatidylinositol PI among the polar lipids, and
Ž .
sterol esters SE in the neutral lipid fraction. After culture with Diet HB Artemia
biomass, the lipid class profile of the octopuses clearly shifted towards the neutral lipids
Ž .
due to an increase in triacylglycerol TAG .
Ž .
The main fatty acids of cephalopods were 16:0, 20:5ny3 and 22:6ny3 Table 1 .
Twenty five to 35% of the total fatty acids were saturated, 9 to 14% were monoenes,
and approximately 50% were polyunsaturated of which the majority were ny3, and
longer than 20 C atoms. Although the PUFA content of all the octopuses analysed was
similar, squid and cuttlefish were richer in ny3 PUFA than octopus, whereas octopus
had higher ny6 PUFA due to relatively high levels of 20:4ny6. Squid paralarvae
were particularly low in ny6 PUFA and thus showed a very high ny3rny6 ratio.
DHA and EPA were also more abundant in squid and cuttlefish hatchlings as compared
to octopus paralarvae.
The lipid class profile of the mysids and pagurid zoeae was clearly distinct from the
Ž .
rest of the food items Fig. 2 . In both groups, the phospholipids were particularly
Ž Ž .
abundant, mainly PC and PE Mysids were also rich in phosphatidylserine PS and
Ž ..
phosphatidic acidrcardiolipin PArCL , whereas in the Artemia diets and pellets, the
neutral lipids were more prominent. HB and SS enriched Artemia were particularly rich
Ž .
in TAG, the former showing also a high proportion of SE. Ethyl esters EE were the
Ž .
most abundant lipid class of pellets together with the free fatty acid fraction FFA .
Chol. was over 10% in all foods. Three distinct fatty acid profiles can be distinguished
among the food items analysed: on one side, mysids and zoeae, on the other, enriched
Ž . Ž .
Artemia, and finally, inert food Table 2 . The natural foods mysids and zoeae were
very rich in PUFA, mainly ny3 fatty acids. Their DHA and EPA content was very
high with the fatty acids in a 1:1 ratio. Pagurid zoeae were richer in ny6 PUFA
compared to the mysids, mainly due to their higher content of arachidonic acid
Ž .
20:4ny6 . The fatty acid profiles of both enriched Artemia were clearly distinguished
by their high content in 18, 20 and 22 C monounsaturated fatty acids, their low levels of
DHA and low DHArEPA ratio. Artemia and pellets were rich in 18:2ny6 in
comparison with mysids and pagurids. The pellets showed higher levels of PUFA than
the Artemia diets, but their fatty acids were richer in monoenes as compared to mysids
and pagurids. Although all food items had a similar proportion of saturated fatty acids
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J.C. NaÕarro, R. VillanueÕarAquaculture 183 2000 161–177 169
Ž . Ž Ž .
Fig. 2. Lipid class composition % of the total lipid of natural food Mysids A. longicornis , Pagurid zoeae
Ž . Ž .
P. prideaux and feeds Artemia SS and HB, Pellets used in the rearing of early stages of cephalopods. Data
are the mean of four replicates. Error bars are the standard deviation. Abbreviations are like in Fig. 1 plus ee:
ethyl esters. For more explanation see the text.
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J.C. NaÕarro, R. VillanueÕarAquaculture 183 2000 161–177
170
Table 2
Ž . Ž .
Total lipid and total fatty acids % of dry weight , and fatty acids from total lipid wt.% of natural food and
feeds used in the rearing of early stages of cephalopods
Data are the means of three replicates. Standard deviations were below 10%.
0.0 are values below 0.09.
Fatty acid Food items
Mysids Pagurid zoeae Artemia SS Artemia HB Pellets
A. longicornis P. prideaux
-14:0 0.8 1.1 0.6 0.4 0.2
14:0 1.4 0.6 1.9 1.5 1.5
14:1 0.2 0.2 0.2 0.3 0.1
15:0 0.8 0.8 0.3 0.4 0.2
15:1 0.2 0.3 0.2 0.0
16:0 22.2 13.8 13.6 12.3 18.4
16:1ny9 0.2 1.0
16:1ny7 1.2 2.6 5.9 4.0 2.3
16:1ny5 0.2 0.2
16:2 0.2 0.5 1.2 0.4
17:0 2.4 0.8 1.0 0.3
16:3 0.3 0.9 0.6 0.6 0.2
16:4 0.1
18:0 4.3 7.8 9.5 8.2 7.4
18:1ny9 5.8 6.8 21.7 20.5 16.7
18:1ny7 4.7 6.2 9.5 8.0 2.0
18:2ny6 1.2 1.2 6.2 7.1 6.3
18:3ny6 0.2 0.1 0.1
18:3ny3 0.5 0.3 3.8 7.0 0.5
18:4ny3 0.5 0.4 0.8 0.8
20:0 0.2 0.4 0.3 0.3 0.3
20:1ny9 0.7 1.0 5.0 4.8 3.3
20:1ny7 0.1 0.9 0.3
20:2ny6 1.7 1.3 0.3 0.4 0.3
20:3ny6 0.2 0.1 0.3
20:4ny6 1.3 5.2 4.0 1.9 2.5
20:3ny3 0.3 0.2 0.2 0.4 0.1
20:4ny3 0.4 0.2 0.2 0.3 0.5
20:5ny3 21.9 15.0 7.1 8.2 12.4
22:0 0.3 0.2 0.1
22:1ny11 0.3 3.0 2.8 2.8
22:1ny9 0.7
22:2ny6 0.3 0.2 0.1 0.6
22:4ny6 0.8 0.3
22:5ny6 0.4 0.8 0.8
22:3ny3 0.1
22:5ny3 0.4 1.8 0.4 0.5 2.1
22:6ny3 24.0 18.1 1.6 2.3 13.4
Total 96.1 91.7 98.0 96.9 98.4
Saturated 28.9 25.9 26.6 23.9 28.0
Monoenes 13.2 18.2 45.3 41.7 28.3
Polyunsaturated 53.2 46.5 25.5 30.9 41.8
ny3 48.1 35.6 13.8 19.4 29.9
ny6 4.8 9.9 10.5 9.7 11.2
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J.C. NaÕarro, R. VillanueÕarAquaculture 183 2000 161–177 171
Ž .
Table 2 continued
Fatty acid Food items
Mysids Pagurid zoeae Artemia SS Artemia HB Pellets
A. longicornis P. prideaux
PUFA ny3 47.1 35.3 9.6 11.7 28.6
PUFA ny6 3.6 8.5 4.3 2.5 4.8
ny3rny6 10.0 3.6 1.3 2.0 2.7
dharepa 1.1 1.2 0.2 0.3 1.1
% total fatty acids 3.0 5.9 7.6 10.7 16.6
% total lipid 12.3 19.2 24.6 37.7 32.9
Ž .
26–29% of total fatty acids , 16:0 was very high in the mysids as compared to all the
other feeds.
After 30 days culture of O. Õulgaris paralarvae, poor growth and survival were
Ž .
achieved Table 3 . During the first 10 days growth was similar in both dietary
treatments, then stopped for 10 more days and increased again to give differences
between the paralarval cultures fed Artemia Diet HB and Artemia Diet SSqpellets.
Ž
Slopes of the growth curves of HB and SS cultures were different Student’s t test,
.
P-0.05 . Better growth was achieved in the HB octopuses but, on the other hand,
Ž .
survival was the lowest Table 3 .
When octopus were cultured for 30 days, their fatty acid composition changed and
Ž .
reflected that of the food Table 1 . The effect of the Artemia diet was clear in the fact
that there was an increase in the content of 18:1 fatty acids, the most abundant fatty acid
in Artemia. Animals fed pellets, richer in DHA than enriched Artemia, showed a higher
proportion of this fatty acid as compared with the other group fed solely by enriched
Artemia Diet HB. O. Õulgaris paralarvae both newly hatched and cultured were very
rich in 20:4ny6 compared with the other cephalopods analysed.
O. Õulgaris paralarvae adopted aiming postures and forward swimming with ex-
tended arms when approaching the pellets, in a behaviour similar to the one observed
Table 3
Ž . Ž .
Mean weight mg and survival % of Octopus Õulgaris at 0, 10, 20 and 30 days of paralarval cultures HB
and SS
Standard deviations in parentheses.
0.0 are values below 0.09.
Days of culture Cultures
Octopus HB Octopus SS
Wet weight Dry weight Survival Wet weight Dry weight Survival
Ž . Ž . Ž . Ž .
0 1.4 0.1 0.3 0.0 100 1.4 0.1 0.3 0.0 100
Ž . Ž . Ž . Ž .
10 1.5 0.3 0.4 0.1 11.0 1.6 0.1 0.4 0.0 33.2
Ž . Ž . Ž . Ž .
20 1.7 0.3 0.5 0.1 4.1 1.7 0.1 0.4 0.0 17.7
Ž . Ž . Ž . Ž .
30 4.4 0.9 1.0 0.2 1.5 3.1 0.3 0.7 0.1 6.7
( )
J.C. NaÕarro, R. VillanueÕarAquaculture 183 2000 161–177
172
Ž .
when they attacked live preys Villanueva and Nozais, 1996 . The pellets were captured
when sinking in the water column. No captures were observed on the pellets deposited
on the bottom of the tank. Rearing observations on the behaviour of pellet capture and
ingestion were done at the age of 15 and 16 days. During six feedings, a minute after the
addition of pellets, 100 individuals were counted, and the number of paralarvae with
pellets within their arms recorded. The mean percentage of paralarvae handling pellets
Ž .
was 49% range: 39–64% . Stomach contents were visualized by transparency, since the
stomach took the pellet colour when ingestion was successful. A total of 62 paralarvae
handling pellets was followed individually by the observer, with 18% of them ingesting
the pellet. This process lasted from 65 to 920 s.
4. Discussion
It has been reported that it is particularly difficult to increase the contents of DHA
Ž
and EPA in enriched Artemia nauplii Dendrinos and Thorpe, 1987; Navarro et al.,
.
1999 . It seems even more difficult to increase the levels of both fatty acids in enriched
Ž .
Artemia biomass in view of the data reported here and elsewhere Naessens et al., 1997 .
The low DHA content in HB and SS enriched Artemia would suggest a deficient
enrichment technique but could also be the result of the lower volume of the digestive
tract-to-body ratio in Artemia biomass as compared to newly hatched nauplii.
All the newly hatched cephalopods showed relatively low lipid contents. Low lipid
contents, with relatively large phospholipid and sterol fractions and scarce reserve lipids
have been hypothesised to be typical of early developmental stages where most of the
Ž .
energy is channelled towards growth Piatkowski and Hagen, 1994 . However, low
Ž
lipids may be a typical common feature of the flesh of many cephalopods Strancari-
.
Stockel and Lozano-Soldevila, 1987 and the higher lipid content in one of the cultured
Ž .
paralarvae Octopus HB can be considered further evidence of a metabolic imbalance
Ž .
caused by improper food composition higher lipid content of diet HB .
Ž
TAG have been reported as minor components of the flesh of cephalopods Jangaard
.
and Ackman, 1965; Nash et al., 1978; Hayashi and Yamamoto, 1987 . They are,
Ž . Ž
however, relatively abundant in the digestive glands of gonatid 3–44% Hayashi and
. Ž . Ž .
Yamamoto, 1987 and ommastrephid squids 58% Nash et al., 1978 . On the other
Ž .
hand, Piatkowski and Hagen 1994 found that TAG represented 13 to 26% of the total
lipid of cranchid squids. This figure agrees with what we report here for newly hatched
O. Õulgaris and for reared paralarvae of the culture SS, but it is higher when compared
with squid and cuttlefish and lower than the values obtained for the HB culture.
Diacylglyceryl ethers and plasmalogens have been described as minor, although
Ž
significant, constituents of several species of cephalopods including the octopus de
Koning, 1972; Hayashi and Kawasaki, 1985; Hayashi and Yamamoto, 1987; Hayashi,
.
1989; Hayashi et al., 1990 . The analytical protocol used in the present work did not
Ž
allow us to separate these constituents from other lipid classes Henderson and Tocher,
.
1992 . Other unusual components, like the ceramide ciliatine, have been described as
Ž .
constituents of the lipids of octopuses de Koning, 1972 . During analysis, these would
( )
J.C. NaÕarro, R. VillanueÕarAquaculture 183 2000 161–177 173
Ž
migrate close to PI and may not have been fully separated Henderson and Tocher,
.
1992 accounting for the high PI of octopus paralarvae.
Ž .
Palmitic acid 16:0 , 20:5ny3 and 22:6ny3 are the most abundant fatty acids
found in the lipids of many cephalopod species, not only at the flesh or whole body level
Ž .
Jangaard and Ackman, 1965; Culkin and Morris, 1970 but also in the digestive glands
Ž . Ž
Jangaard and Ackman, 1965; Hayashi et al., 1990 , the central nervous system Dumont
. Ž .
et al., 1992; Dumont et al., 1994 and in photoreceptors Eguchi et al., 1994 . In view of
this fatty acid composition and the data reported here, one can deduce that the long
chain PUFA requirements of cephalopod paralarvae must be very high.
There was a clear influence of the lipid composition of the food on the lipids of
cultured octopus paralarvae both at the level of lipid class and fatty acid composition.
HB Artemia fed octopuses showed a clear increase in TAG, whereas the octopus
paralarvae fed pellets showed a lipid class profile closer to the one of the newly hatched
individuals. The paralarvae fed pellets had a higher content of free fatty acids, which
were abundant, together with ethyl esters, in the pellets. The influence of the lipid
composition of the natural food on the body composition of cephalopods both in the
Ž .
flesh and in the digestive gland has also been discussed by other authors Joseph, 1989 .
The dietary requirements for ny3 PUFA, particularly DHA is critical in early
developmental stages due to the high demand for membrane synthesis where the ny3
Ž .
PUFA are incorporated Henderson and Sargent, 1985 . DHA plays an important role in
Ž
maintaining the structural and functional integrity of cell membranes in fish Sargent,
.
1995 . This fatty acid may be even more important for the correct development and
survival of fast growing phospholipid-rich cephalopod paralarvae.
The cultured octopus paralarvae showed a lower content of PUFA as compared with
the newly hatched individuals. The differences were particularly marked with DHA and
EPA, were probably of dietary aetiology in view of the results obtained when fed
pelleted diet, and can be the cause of the poor performance of the paralarval rearing.
Fish larvae are strongly dependent on the fatty acids of their prey for the elaboration
Ž
of major polar lipids in key organs including neural and visual apparatus Navarro et al.,
. Ž .
1993b, 1995 . Navarro et al. 1993b reported that 30 days of dietary manipulation were
sufficient to change the fatty acid composition of the lipids of the heads, eyes and bodies
Ž
of herring larvae. Similar results were obtained with sea bass larvae Navarro et al.,
. Ž . Ž
1995 and with weaned turbot Mourente et al., 1991 and sea bream Mourente and
.
Tocher, 1993 . In view of the data reported here, this dietary effect seems to be a fast
Ž .
generalised process not limited to fish and crustacean Montano and Navarro, 1996
˜
Ž .
larvae, whose physical poor growth, survival and resistance and physiological signifi-
Ž .
cance Bell et al., 1995 can perhaps be extrapolated to cephalopod paralarvae.
Despite the better lipid class profile and fatty acid composition of group SS, low
survival and growth were reached. Since the lower survival of this group was obtained
from the beginning of the culture, causes other than the quality of food may be directly
involved. In fact, the clogging of the filters at day 8 and a decrease in water quality due
to an excess of pellets offered to the paralarvae at day 10 seem to be the causes of peak
moralities in cultures HB and SS, respectively. Aside from these considerations, both
cultures gave poor growth and survival as compared to other cultures carried out using
Ž .
crustacean zoeae as food see Villanueva, 1995 , pointing to causes other than simply
( )
J.C. NaÕarro, R. VillanueÕarAquaculture 183 2000 161–177
174
the lipid composition of food for the lack of success in this paralarval culture, perhaps a
strong dietary imbalance of the enriched Artemia and difficulties in getting the
paralarvae to accept inert food.
During the paralarval rearing, it was observed that the paralarvae fed on the pelleted
diet. With our experimental design it was impossible to quantify the amount of inert
food ingested but it is clear that this had an effect on the lipid composition of paralarvae,
which encourages the use of co-feeding techniques to correct the deficient nutrient
Ž .
composition of Artemia as sole paralarval food Rosenlund et al., 1997 . This is the first
time that pellets have been used for rearing paralarval stages of cephalopods. In contrast
with larval fishes, cephalopod paralarvae manipulate the food with their arms before
ingestion after capture. The high incidence of captured pellets by O. Õulgaris paralarvae
but with subsequent low ingestion indicates that palatability could be a determinant
factor on the ingestion rates. Although during the present study, low success was
obtained with pellet ingestion, co-feeding techniques can be a way in the future for
rearing cephalopod paralarvae, provided palatability and feeding protocols are improved.
Pelleted diets have been previously used for rearing subadult stages, indicating the
Ž
adaptability of cephalopods to feed on inert food Lee et al., 1991; Castro et al., 1993;
.
Castro and Lee, 1994 .
5. Conclusion
In view of the present results, Artemia does not seem to be the right choice to feed
early stages of cephalopods. From their lipid composition and that of their natural food,
it can be deduced that cephalopod paralarvae and juveniles must require a food rich in
PUFA, phopholipids and cholesterol and with a moderate content of neutral lipids. This
is particularly clear for the squid L. Õulgaris and the cuttlefish S. officinalis. However,
these theoretical lipid requirements are common to most fish and crustacean larvae that,
on the other hand, are reared with different degrees of success using enriched Artemia
nauplii. Fine tuning of enrichment by using enrichment diets high in polar lipids
Ž .
McEvoy et al., 1996 and PUFA andror co-feeding enriched Artemia with inert diets
similar to the pellets used in the present experiments should be the choice to attempt
paralarval rearing of cephalopods with minimum guarantees of success.
Acknowledgements
We appreciate the technical assistance of Mr. J. Riba during the rearing experiences.
We are grateful to Miguel A. Montolio and M. Angeles G. Albaladejo who helped in the
Ž .
lipid analysis. Mr. G. Munoz-Ramos Escola del Mar de Badalona collected the
˜
mysidiacean shrimps. This study was partially funded by a joint research project
between the CSIC and the ‘‘Department d’Agricultura, Ramaderia i Pesca’’ of the
Catalan Government.
( )
J.C. NaÕarro, R. VillanueÕarAquaculture 183 2000 161–177 175
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  • 1. Ž . Aquaculture 183 2000 161–177 www.elsevier.nlrlocateraqua-online Lipid and fatty acid composition of early stages of cephalopods: an approach to their lipid requirements Juan C. Navarro a,) , Roger Villanueva b a ( ) Instituto de Acuicultura de Torre de la Sal CSIC , E-12595 Ribera de Cabanes, Castellon, Spain ´ b ( ) Instituto de Ciencias del Mar CSIC , Paseo Juan de Borbon srn, E-08039 Barcelona, Spain ´ Accepted 10 August 1999 Abstract We report here the main lipid classes and the fatty acid composition from the total lipids of hatchlings of three cephalopod species: Sepia officinalis, Loligo Õulgaris and Octopus Õulgaris, as well as the lipid composition of two selected crustaceans that have been used previously with success as food resource for rearing cephalopod hatchlings: zoeae of Pagurus prideaux and the mysidacean Acanthomysis longicornis. Additionally, we report the lipid class and fatty acid composition of two cultures of O. Õulgaris paralarvae reared with enriched Artemia juveniles, and with enriched Artemia juveniles plus a prepared pelleted diet. From their lipid composition and that of their natural food, it can be deduced that cephalopod paralarvae and juveniles must Ž . require a food rich in polyunsaturated fatty acids PUFA , phospholipids and cholesterol and with a moderate content in neutral lipids. There was a clear influence of the lipid composition of the food on the lipids of cultured octopus paralarvae both at the level of lipid class and fatty acid composition. The cultured octopus paralarvae showed a lower content of PUFA as compared with the newly hatched individuals. Co-feeding techniques based on the use of polar lipid and PUFA enriched Artemia together with palatable pellets seemed to be a possible way to improve paralarval and juvenile cephalopod culture beyond the experimental scale. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Lipids; Fatty acids; Cephalopods; Octopus; Squid; Cuttlefish ) Corresponding author. Tel.: q34-964319500; fax: q34-964319509; e-mail: jcnavarro@iats.csic.es 0044-8486r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. Ž . PII: S0044-8486 99 00290-2
  • 2. ( ) J.C. NaÕarro, R. VillanueÕarAquaculture 183 2000 161–177 162 1. Introduction Cephalopods have been cultured through the complete life cycle and multiple generations only at an experimental scale or, in few numbers for biomedical research. Ž The cuttlefish Sepia officinalis Pascual, 1978; Boletzky, 1983; Forsythe et al., 1994; . Ž . Hanley et al., 1998 and Sepioteuthis lessoniana Lee et al., 1994 seem to be the most easily cultured due, among other reasons, to the large size of the hatchlings. Many cephalopod species, mostly squids and octopods, have a planktonic posthatching stage, Ž . and the term paralarva has been proposed for these stages Young and Harman, 1988 . In other cephalopods, such as the cuttlefishes, hatchlings are large and immediately benthic like the adults and thus are referred to as juveniles. Paralarvae and juveniles are active swimmers and rapid growing forms that require substantial amounts of food Ž . Vecchione, 1991; Villanueva and Nozais, 1996; Villanueva et al., 1995 . In fact, subadult and adult cephalopods show high metabolic rates and fast growth which are Ž . sustained by high feeding rates O’Dor and Wells, 1987 . Digestive capability of Ž . paralarval stages Boucaud-Camou and Roper, 1995, 1998 indicate a carnivorous diet Ž . Ž . as in juveniles Boucaud-Camou et al., 1985 and adults Boucher-Rodoni et al., 1987 . The culture of cephalopods is becoming an important area of interest due to their Ž y1 . Ž . rapid growth )5% body weight day Forsythe and Van Heukelem, 1987 and high market price. A suitable species is the common octopus Octopus Õulgaris, due to its Ž . rapid growth and high food conversion Mangold and Boletzky, 1973; Mangold, 1983 . At present, the commercial culture of O. Õulgaris in Spanish waters is limited to Ž ongrowing subadults captured from the wild, to adult sizes Iglesias et al., 1997; . Rama-Villar et al., 1997 . Octopus with planktonic stages like O. Õulgaris, have been Ž successfully reared through their paralarval stages only at an experimental scale Itami et . al., 1963; Villanueva, 1995 . The paralarval culture of octopus and early stages of most cephalopod species, still being a bottleneck. The suitable food for rearing these early stages has been an unresolved problem. At present, most of the live prey used for successfully rearing early stages are collected from the sea, as zooplankton for paralarval Ž . squids Yang et al., 1986; Hanlon et al., 1979, 1989 or mysids and palaemonids for Ž . juvenile cuttlefishes and squids Forsythe et al., 1994; Lee, 1994 , but this method requires a substantial input of time and effort. On the other hand, laboratory hatched Ž decapod zoeae have been used as food for paralarval octopods and squids Itami et al., . 1963; Villanueva, 1994, 1995 , but the use of this food item requires a risky cumber- some parallel culture of crustaceans that seems impractical beyond the experimental scale systems. Aside from the problems related to food size and quantity, there seem to be other problems associated with food quality. Since cephalopods have a predominant amino Ž . acid metabolism Lee, 1994 , research on the lipid and fatty acid requirements have been somewhat neglected to the point that these are scarcely known at present. Adult Ž . Ž cephalopods are rich in long chain polyunsaturated fatty acids PUFA Jangaard and . Ackman, 1965; Nash et al., 1978 , their oil, being a good source of these compounds Ž often used in aquaculture to supplement feeds Southgate and Lou, 1995; Knauer and . Southgate, 1997 . As far as we are aware, there are no reports on the fatty acid composition of paralarval and juvenile cephalopods. According to the fatty acid compo-
  • 3. ( ) J.C. NaÕarro, R. VillanueÕarAquaculture 183 2000 161–177 163 sition of the adults, and to the generic composition of their natural food, the paralarvae must require high amounts of these fatty acids. This hypothesis is further substantiated by the fact that earlier attempts to culture octopus and squid paralarvae with the brine Ž . shrimp Artemia failed Boletzky and Hanlon, 1983; Hamazaki et al., 1991 . It is known that Artemia, in general, lacks long chain PUFA essential for marine animals, like Ž . eicosapentaenoic acid EPA, 20:5ny3 which may be present in low amounts or absent Ž . in the lipid of the brine shrimp, and docosahexaenoic acid DHA, 22:6ny3 which is Ž . basically absent Navarro et al., 1992, 1993a . The aim of the present work was to take a first insight on the nutritional requirements of the paralarval and juvenile stages of cephalopods in culture. First, we determined the main lipid classes and the fatty acid composition of the newly hatched individuals of three cephalopod species that represent the three main cephalopod orders, all of them of high commercial interest: the cuttlefish S. officinalis, the European squid L. Õulgaris and the common octopus O. Õulgaris. Second, we analysed the same lipid composition of two selected crustaceans that have been successfully used previously as food resources for rearing cephalopod hatchlings, under experimental cultures: zoeae of the hermit crab Pagurus prideaux and the mysidacean shrimp Acanthomysis longicornis Ž . Villanueva, 1994 . Finally, we report the lipid class and fatty acid composition of two cultures of O. Õulgaris paralarvae reared with enriched Artemia juveniles, and with enriched Artemia juveniles plus a prepared pelleted diet. 2. Materials and methods 2.1. Collection of material Ž Cephalopod eggs were collected off Vilanova i la Geltru, Barcelona NW Mediter- ´ . ranean by means of the local ceramic pot fishery for octopus. The pot line was placed near the coast, between 10 and 25 m depth. Egg masses of S. officinalis and egg capsules of L. Õulgaris, attached to the fishery line were collected on board and Ž . transported to the lab the same day February 1998 . Eggs were incubated at ambient Ž . temperature mean: 15.28C, range: 14.7–15.68C , using an open-circuit of filtered sea water at the ICM, Barcelona. During February and March 1998, active, healthy individuals of the three cephalopod species: S. officinalis, L. Õulgaris and O. Õulgaris, recently hatched in aquaria were collected for analysis. Additionally, two crustacean species were analyzed for lipid and fatty acid composi- tion. Ovigerous females of the hermit crab P. prideaux were collected from the by-catch of the craft fishery from the same place and depth as above, in January 1998. Ovigerous Ž . females were transported to the lab, maintained as described in Villanueva 1994 and Ž . the recent hatched zoeae used for analysis. Mysids A. longicornis were collected in May 1998, off Badalona, Barcelona, by a hand net, at 10 m depth using scuba diving. The same day the individuals were preserved for analysis. Freshly hatched individuals of cephalopods and crustaceans were collected and preserved during the first 24 h after hatching in aquaria. The samples were collected using a hand net, washed in freshwater, then placed in blotting paper to remove the
  • 4. ( ) J.C. NaÕarro, R. VillanueÕarAquaculture 183 2000 161–177 164 water, and weighed using an Ohaus Analytical Plus AP250D-O microbalance. Samples Ž . were preserved in chloroformrmethanol 2:1, vrv with 0.01% butylated hydroxy- Ž . toluene wrv as antioxidant. From each sample three aliquot replicates were used for dry weight determinations after drying in an oven for 48 h at 1008C. 2.2. Rearing experiments of O. Õulgaris paralarÕae O. Õulgaris broodstock collected from local craft fishery was maintained using the Ž . open-circuit seawater system of the ICM. Females 1–2 kg were placed with males of similar size during 2–3 days to ensure mating. Fertilised females were isolated and maintained in 200-l individual cylindrical plastic tanks. An overabundance of food was Ž . Ž . supplied using frozen crabs Carcinus maenas and sardine Sardina pilchardus . All fertilised females were maintained in the dark with the aim of accelerating their sexual Ž . maturation Wells, 1978; Zuniga et al., 1995 . Some of them were also kept at warm ´˜ Ž . temperature by heating the water to 19–218C with the same purpose Villanueva, 1995 . Embryonic development of the egg masses and hatching time were controlled according to the rearing needs by means of the temperature of the water, since this is the main Ž . factor that modulates the duration of the embryonic period Boletzky, 1989 . A 700-l semi-closed seawater system connected to the open-circuit of the ICM was used for the paralarval rearing experiments. Seawater was filtered through 0.2 mm and daily renovation rates were 10–40%. The semi-closed system was equipped with a biological filter, protein skimmer, UV lamps, ozonizer and temperature control. Two rearing experiments of O. Õulgaris from hatching to 30 days were carried out. Artemia biomass and pellets were used as food. Ž . Artemia AF, Artemia Systems nauplii were grown in seawater at 25–308C under constant illumination during 7–10 days using 45-l cylindro-conical plastic tanks. Artemia cultures were fed Dunaliella Õiridis algae to obtain Artemia biomass composed of 1–3 mm total length individuals. This prey size was selected because it had been previously Ž . used with success for rearing O. Õulgaris paralarvae Villanueva, 1994, 1995 . The Artemia biomass was enriched in seawater for 24 h at 288C with one of the following Ž . Ž . y1 Ž . enrichment diets: a Diet SS: DC Super Selco Artemia Systems 0.6 g l and, b Diet y1 Ž . y1 HB: DC Super Selco 0.6 g l , cod liver oil Acofarma Laboratories 0.6 g l , vitamin Ž . y1 Ž . y1 C Sigma Products 0.3 g l , Hipramin-B Hipra Laboratories 0.4 ml l . Fifty y1 y1 Ž . thousand IU l of sodium G-penicillin and 50 mg l streptomycin sulphate Sigma were added to both enrichment media to retard bacterial growth. Ž . The composition and formulation of the pelleted diet in dry weight % was: frozen Ž . Ž . Ž . euphausiids MBF 30%, squid Todarodes sagittatus powder Rieber and Son 18%, Ž . Ž . fish hydrolysate Divaq 18%, DC Super Selco 8%, phosphatidylcholine PC, Sigma Ž . Ž . Ž 5%, cholesterol Sigma 3%, vitamin complex Kurios 5%, mineral complex Warner . Ž . Ž . Lambert 5%, gelatine Royal 7.975%, ethoxyquin Sigma 0.025%. Ingredients were mixed with an electric blender, pelletized and dried at room temperature during 48 h. The percentage of moisture was 6%. The pellets were sieved to obtain particle size of 250–500 mm and were stored at y208C before use. The colour of the pellet was reddish brown.
  • 5. ( ) J.C. NaÕarro, R. VillanueÕarAquaculture 183 2000 161–177 165 Ž . Fig. 1. Lipid class composition % of the total lipid of early stages of cephalopods: lpc: lysophosphatidyl- choline, sm: shingomyelin, pc: phosphatidylcholine, ps: phosphatidylserine, pi: phosphatidylinositol, parcl: phosphatidic acidrcardiolipin, pe: phosphatidylethanolamine, pigm: pigments, magrdag: monordi acylglyc- erides, chol: cholesterol, tag: triacylglycerides, ffa: free fatty acids, se: sterol esters. Data are the mean of four replicates. Error bars are the standard deviation.
  • 6. ( ) J.C. NaÕarro, R. VillanueÕarAquaculture 183 2000 161–177 166 Table 1 Ž . Ž . Total lipid and total fatty acids % of dry weight , and fatty acids from total lipids wt.% of early stages of cephalopods Data are the means of three replicates. Standard deviations were below 10%. 0.0 are values below 0.09. Fatty acid Hatchlings Paralarval culture O. Õulgaris S. officinalis L. Õulgaris Octopus HB Octopus SS -14:0 1.0 0.2 0.4 1.6 0.8 14:0 1.8 2.7 2.1 0.6 0.5 14:1 0.2 0.2 15:0 0.2 0.2 0.5 0.2 0.2 15:1 0.1 16:0 17.5 22.3 22.1 12.1 15.7 16:1ny9 0.7 0.1 0.3 0.2 16:1ny7 0.5 0.2 0.2 1.5 0.7 16:1ny5 0.4 16:2 0.1 0.2 0.2 17:0 1.4 1.0 1.7 1.4 1.2 16:3 0.1 0.1 0.2 0.3 0.3 16:4 0.1 0.7 0.1 18:0 6.3 8.7 9.1 11.4 12.2 18:1ny11 1.4 0.4 0.5 0.7 18:1ny9 4.0 4.0 2.8 11.7 5.3 18:1ny7 1.6 1.1 1.3 6.7 3.9 18:1ny5 0.2 18:2ny6 3.3 0.4 0.3 2.9 1.9 18:3ny6 0.1 18:3ny3 2.1 1.0 18:4ny3 0.1 20:0 0.1 0.2 0.3 0.3 0.2 20:1ny9 4.6 5.4 3.5 4.0 2.7 20:1ny7 0.4 0.3 0.3 20:2ny6 0.9 0.5 0.4 1.1 0.9 20:3ny6 0.2 20:4ny6 7.3 0.9 3.3 4.9 7.3 20:3ny3 0.7 0.4 0.6 1.2 1.2 20:4ny3 0.3 0.1 20:5ny3 12.6 14.3 14.5 17.4 14.4 22:0 0.1 22:1ny11 0.6 0.1 0.1 0.4 0.3 22:1ny9 0.5 0.4 22:2ny6 0.1 0.1 22:4ny6 0.9 0.2 0.2 22:5ny6 0.5 0.5 0.6 0.2 0.3 22:5ny3 1.7 0.5 1.1 1.5 1.0 22:6ny3 21.2 32.8 29.3 9.8 19.1 Total 90.9 96.6 96.2 95.0 94.3 Saturated 27.2 35.0 35.7 25.9 30.1 Monoenes 13.5 11.1 9.0 25.6 14.8 Polyunsaturated 49.2 50.3 51.1 41.9 48.6 ny3 36.2 48.0 45.5 32.5 37.0 ny6 12.9 2.1 4.7 8.9 11.1
  • 7. ( ) J.C. NaÕarro, R. VillanueÕarAquaculture 183 2000 161–177 167 Ž . Table 1 continued Fatty acid Hatchlings Paralarval culture O. Õulgaris S. officinalis L. Õulgaris Octopus HB Octopus SS PUFA ny3 36.2 48.0 45.5 30.2 35.9 PUFA ny6 9.6 1.7 4.5 6.0 9.1 ny3rny6 2.9 23.4 9.6 3.6 3.3 dharepa 1.7 2.3 2.0 0.6 1.3 % total fatty acids 4.6 4.6 4.1 7.2 5.4 % total lipid 13.4 12.5 10.8 25.1 13.7 2.2.1. O. Õulgaris paralarÕal culture HB The rearing experiment started with a total of 1250 freshly hatched O. Õulgaris paralarvae divided in two cylindric polyethylene plastic tanks. The characteristics of Ž these tanks and general rearing conditions have been described elsewhere Villanueva, . y1 1995 . Tank volume was 70 l and water flow 120 l h . Temperature ranged from 20 to Ž . Ž . 228C. Artemia biomass Diet HB was supplied ad libitum 2–3 times a day from the first day of culture to the end at 30 days. 2.2.2. O. Õulgaris paralarÕal culture SS The experiment started with a total of 1600 freshly hatched O. Õulgaris paralarvae divided in four cylindric PVC tanks. Tank volume was 25 l and water flow 80 l hy1 . Ž . Temperature ranged from 20.6 to 22.58C. As feeding regime Artemia biomass Diet SS Ž . was supplied ad libitum 2–3 times a day through the experimental period and pelleted diet from day 10 to 30. Pellets were continuously supplied in excess 24 h dayy1 using Ž . an automatic delivery system Dohse Aquaristik . In both experiments illumination was constant 24 h dayy1 . Tanks were cleaned daily and dead animals removed and counted. At days 10, 20 and 30, samples of 15 paralarvae Ž . were cold-anaesthetised 2–48C and weighed after blotting on paper to remove the water of the mantle cavity. Dry weights were obtained after drying the samples in an oven for 48 h at 1008C. 2.3. Analytical Ž . Lipids were extracted using the method of Folch et al. 1957 . Lipid classes were separated by high performance thin layer chromatography using the method of Olsen Ž . Ž and Henderson 1989 , and quantified by densitometry Bio Rad GS 670 Imaging . Ž densitometer . For fatty acid analyses, aliquots were transmethylated overnight Christie, . Ž . 1982 after the addition of 19:0 as an internal standard Sigma . Methyl esters were Ž . extracted with hexanerdiethyl ether 1:1, vrv , and purified by thin layer chromatogra- Ž . Ž phy Silica Gel G 60, Merck using hexanerdiethyl etherracetic acid 85:15:1.5, . vrvrv as solvent. The analyses of the methyl esters were carried out in a Fisons 8000 gas chromatograph equipped with a fused silica 30 m=0.25 mm open tubular column Ž . Tracer, TR-WAX, film thickness: 0.25 mm and a cold on-column injection system, using helium as carrier gas, and a thermal gradient from 50 to 2208C. Peaks were
  • 8. ( ) J.C. NaÕarro, R. VillanueÕarAquaculture 183 2000 161–177 168 recorded in a personal computer linked to the analytical instrument using Chrom-Card Ž . software Fisons , quantified with the aid of the response factor of the internal standard and identified by comparison with a well characterised marine oil. 3. Results All the cephalopod paralarvae and juveniles were rich in polar lipid and cholesterol Ž . Ž . Chol. , the latter ranging from 18 to 25% of total lipid Fig. 1 . In general, PC, Ž . phosphatidylethanolamine PE and Chol. accounted for more than 60% of the total Ž . Ž . lipid. Squid and cuttlefish were richer in PC 34, 33% and PE 25, 22% as compared to Ž . newly hatched octopuses 21, 16% . The lipids of O. Õulgaris paralarvae, both newly Ž . hatched and cultured were richer in phosphatidylinositol PI among the polar lipids, and Ž . sterol esters SE in the neutral lipid fraction. After culture with Diet HB Artemia biomass, the lipid class profile of the octopuses clearly shifted towards the neutral lipids Ž . due to an increase in triacylglycerol TAG . Ž . The main fatty acids of cephalopods were 16:0, 20:5ny3 and 22:6ny3 Table 1 . Twenty five to 35% of the total fatty acids were saturated, 9 to 14% were monoenes, and approximately 50% were polyunsaturated of which the majority were ny3, and longer than 20 C atoms. Although the PUFA content of all the octopuses analysed was similar, squid and cuttlefish were richer in ny3 PUFA than octopus, whereas octopus had higher ny6 PUFA due to relatively high levels of 20:4ny6. Squid paralarvae were particularly low in ny6 PUFA and thus showed a very high ny3rny6 ratio. DHA and EPA were also more abundant in squid and cuttlefish hatchlings as compared to octopus paralarvae. The lipid class profile of the mysids and pagurid zoeae was clearly distinct from the Ž . rest of the food items Fig. 2 . In both groups, the phospholipids were particularly Ž Ž . abundant, mainly PC and PE Mysids were also rich in phosphatidylserine PS and Ž .. phosphatidic acidrcardiolipin PArCL , whereas in the Artemia diets and pellets, the neutral lipids were more prominent. HB and SS enriched Artemia were particularly rich Ž . in TAG, the former showing also a high proportion of SE. Ethyl esters EE were the Ž . most abundant lipid class of pellets together with the free fatty acid fraction FFA . Chol. was over 10% in all foods. Three distinct fatty acid profiles can be distinguished among the food items analysed: on one side, mysids and zoeae, on the other, enriched Ž . Ž . Artemia, and finally, inert food Table 2 . The natural foods mysids and zoeae were very rich in PUFA, mainly ny3 fatty acids. Their DHA and EPA content was very high with the fatty acids in a 1:1 ratio. Pagurid zoeae were richer in ny6 PUFA compared to the mysids, mainly due to their higher content of arachidonic acid Ž . 20:4ny6 . The fatty acid profiles of both enriched Artemia were clearly distinguished by their high content in 18, 20 and 22 C monounsaturated fatty acids, their low levels of DHA and low DHArEPA ratio. Artemia and pellets were rich in 18:2ny6 in comparison with mysids and pagurids. The pellets showed higher levels of PUFA than the Artemia diets, but their fatty acids were richer in monoenes as compared to mysids and pagurids. Although all food items had a similar proportion of saturated fatty acids
  • 9. ( ) J.C. NaÕarro, R. VillanueÕarAquaculture 183 2000 161–177 169 Ž . Ž Ž . Fig. 2. Lipid class composition % of the total lipid of natural food Mysids A. longicornis , Pagurid zoeae Ž . Ž . P. prideaux and feeds Artemia SS and HB, Pellets used in the rearing of early stages of cephalopods. Data are the mean of four replicates. Error bars are the standard deviation. Abbreviations are like in Fig. 1 plus ee: ethyl esters. For more explanation see the text.
  • 10. ( ) J.C. NaÕarro, R. VillanueÕarAquaculture 183 2000 161–177 170 Table 2 Ž . Ž . Total lipid and total fatty acids % of dry weight , and fatty acids from total lipid wt.% of natural food and feeds used in the rearing of early stages of cephalopods Data are the means of three replicates. Standard deviations were below 10%. 0.0 are values below 0.09. Fatty acid Food items Mysids Pagurid zoeae Artemia SS Artemia HB Pellets A. longicornis P. prideaux -14:0 0.8 1.1 0.6 0.4 0.2 14:0 1.4 0.6 1.9 1.5 1.5 14:1 0.2 0.2 0.2 0.3 0.1 15:0 0.8 0.8 0.3 0.4 0.2 15:1 0.2 0.3 0.2 0.0 16:0 22.2 13.8 13.6 12.3 18.4 16:1ny9 0.2 1.0 16:1ny7 1.2 2.6 5.9 4.0 2.3 16:1ny5 0.2 0.2 16:2 0.2 0.5 1.2 0.4 17:0 2.4 0.8 1.0 0.3 16:3 0.3 0.9 0.6 0.6 0.2 16:4 0.1 18:0 4.3 7.8 9.5 8.2 7.4 18:1ny9 5.8 6.8 21.7 20.5 16.7 18:1ny7 4.7 6.2 9.5 8.0 2.0 18:2ny6 1.2 1.2 6.2 7.1 6.3 18:3ny6 0.2 0.1 0.1 18:3ny3 0.5 0.3 3.8 7.0 0.5 18:4ny3 0.5 0.4 0.8 0.8 20:0 0.2 0.4 0.3 0.3 0.3 20:1ny9 0.7 1.0 5.0 4.8 3.3 20:1ny7 0.1 0.9 0.3 20:2ny6 1.7 1.3 0.3 0.4 0.3 20:3ny6 0.2 0.1 0.3 20:4ny6 1.3 5.2 4.0 1.9 2.5 20:3ny3 0.3 0.2 0.2 0.4 0.1 20:4ny3 0.4 0.2 0.2 0.3 0.5 20:5ny3 21.9 15.0 7.1 8.2 12.4 22:0 0.3 0.2 0.1 22:1ny11 0.3 3.0 2.8 2.8 22:1ny9 0.7 22:2ny6 0.3 0.2 0.1 0.6 22:4ny6 0.8 0.3 22:5ny6 0.4 0.8 0.8 22:3ny3 0.1 22:5ny3 0.4 1.8 0.4 0.5 2.1 22:6ny3 24.0 18.1 1.6 2.3 13.4 Total 96.1 91.7 98.0 96.9 98.4 Saturated 28.9 25.9 26.6 23.9 28.0 Monoenes 13.2 18.2 45.3 41.7 28.3 Polyunsaturated 53.2 46.5 25.5 30.9 41.8 ny3 48.1 35.6 13.8 19.4 29.9 ny6 4.8 9.9 10.5 9.7 11.2
  • 11. ( ) J.C. NaÕarro, R. VillanueÕarAquaculture 183 2000 161–177 171 Ž . Table 2 continued Fatty acid Food items Mysids Pagurid zoeae Artemia SS Artemia HB Pellets A. longicornis P. prideaux PUFA ny3 47.1 35.3 9.6 11.7 28.6 PUFA ny6 3.6 8.5 4.3 2.5 4.8 ny3rny6 10.0 3.6 1.3 2.0 2.7 dharepa 1.1 1.2 0.2 0.3 1.1 % total fatty acids 3.0 5.9 7.6 10.7 16.6 % total lipid 12.3 19.2 24.6 37.7 32.9 Ž . 26–29% of total fatty acids , 16:0 was very high in the mysids as compared to all the other feeds. After 30 days culture of O. Õulgaris paralarvae, poor growth and survival were Ž . achieved Table 3 . During the first 10 days growth was similar in both dietary treatments, then stopped for 10 more days and increased again to give differences between the paralarval cultures fed Artemia Diet HB and Artemia Diet SSqpellets. Ž Slopes of the growth curves of HB and SS cultures were different Student’s t test, . P-0.05 . Better growth was achieved in the HB octopuses but, on the other hand, Ž . survival was the lowest Table 3 . When octopus were cultured for 30 days, their fatty acid composition changed and Ž . reflected that of the food Table 1 . The effect of the Artemia diet was clear in the fact that there was an increase in the content of 18:1 fatty acids, the most abundant fatty acid in Artemia. Animals fed pellets, richer in DHA than enriched Artemia, showed a higher proportion of this fatty acid as compared with the other group fed solely by enriched Artemia Diet HB. O. Õulgaris paralarvae both newly hatched and cultured were very rich in 20:4ny6 compared with the other cephalopods analysed. O. Õulgaris paralarvae adopted aiming postures and forward swimming with ex- tended arms when approaching the pellets, in a behaviour similar to the one observed Table 3 Ž . Ž . Mean weight mg and survival % of Octopus Õulgaris at 0, 10, 20 and 30 days of paralarval cultures HB and SS Standard deviations in parentheses. 0.0 are values below 0.09. Days of culture Cultures Octopus HB Octopus SS Wet weight Dry weight Survival Wet weight Dry weight Survival Ž . Ž . Ž . Ž . 0 1.4 0.1 0.3 0.0 100 1.4 0.1 0.3 0.0 100 Ž . Ž . Ž . Ž . 10 1.5 0.3 0.4 0.1 11.0 1.6 0.1 0.4 0.0 33.2 Ž . Ž . Ž . Ž . 20 1.7 0.3 0.5 0.1 4.1 1.7 0.1 0.4 0.0 17.7 Ž . Ž . Ž . Ž . 30 4.4 0.9 1.0 0.2 1.5 3.1 0.3 0.7 0.1 6.7
  • 12. ( ) J.C. NaÕarro, R. VillanueÕarAquaculture 183 2000 161–177 172 Ž . when they attacked live preys Villanueva and Nozais, 1996 . The pellets were captured when sinking in the water column. No captures were observed on the pellets deposited on the bottom of the tank. Rearing observations on the behaviour of pellet capture and ingestion were done at the age of 15 and 16 days. During six feedings, a minute after the addition of pellets, 100 individuals were counted, and the number of paralarvae with pellets within their arms recorded. The mean percentage of paralarvae handling pellets Ž . was 49% range: 39–64% . Stomach contents were visualized by transparency, since the stomach took the pellet colour when ingestion was successful. A total of 62 paralarvae handling pellets was followed individually by the observer, with 18% of them ingesting the pellet. This process lasted from 65 to 920 s. 4. Discussion It has been reported that it is particularly difficult to increase the contents of DHA Ž and EPA in enriched Artemia nauplii Dendrinos and Thorpe, 1987; Navarro et al., . 1999 . It seems even more difficult to increase the levels of both fatty acids in enriched Ž . Artemia biomass in view of the data reported here and elsewhere Naessens et al., 1997 . The low DHA content in HB and SS enriched Artemia would suggest a deficient enrichment technique but could also be the result of the lower volume of the digestive tract-to-body ratio in Artemia biomass as compared to newly hatched nauplii. All the newly hatched cephalopods showed relatively low lipid contents. Low lipid contents, with relatively large phospholipid and sterol fractions and scarce reserve lipids have been hypothesised to be typical of early developmental stages where most of the Ž . energy is channelled towards growth Piatkowski and Hagen, 1994 . However, low Ž lipids may be a typical common feature of the flesh of many cephalopods Strancari- . Stockel and Lozano-Soldevila, 1987 and the higher lipid content in one of the cultured Ž . paralarvae Octopus HB can be considered further evidence of a metabolic imbalance Ž . caused by improper food composition higher lipid content of diet HB . Ž TAG have been reported as minor components of the flesh of cephalopods Jangaard . and Ackman, 1965; Nash et al., 1978; Hayashi and Yamamoto, 1987 . They are, Ž . Ž however, relatively abundant in the digestive glands of gonatid 3–44% Hayashi and . Ž . Ž . Yamamoto, 1987 and ommastrephid squids 58% Nash et al., 1978 . On the other Ž . hand, Piatkowski and Hagen 1994 found that TAG represented 13 to 26% of the total lipid of cranchid squids. This figure agrees with what we report here for newly hatched O. Õulgaris and for reared paralarvae of the culture SS, but it is higher when compared with squid and cuttlefish and lower than the values obtained for the HB culture. Diacylglyceryl ethers and plasmalogens have been described as minor, although Ž significant, constituents of several species of cephalopods including the octopus de Koning, 1972; Hayashi and Kawasaki, 1985; Hayashi and Yamamoto, 1987; Hayashi, . 1989; Hayashi et al., 1990 . The analytical protocol used in the present work did not Ž allow us to separate these constituents from other lipid classes Henderson and Tocher, . 1992 . Other unusual components, like the ceramide ciliatine, have been described as Ž . constituents of the lipids of octopuses de Koning, 1972 . During analysis, these would
  • 13. ( ) J.C. NaÕarro, R. VillanueÕarAquaculture 183 2000 161–177 173 Ž migrate close to PI and may not have been fully separated Henderson and Tocher, . 1992 accounting for the high PI of octopus paralarvae. Ž . Palmitic acid 16:0 , 20:5ny3 and 22:6ny3 are the most abundant fatty acids found in the lipids of many cephalopod species, not only at the flesh or whole body level Ž . Jangaard and Ackman, 1965; Culkin and Morris, 1970 but also in the digestive glands Ž . Ž Jangaard and Ackman, 1965; Hayashi et al., 1990 , the central nervous system Dumont . Ž . et al., 1992; Dumont et al., 1994 and in photoreceptors Eguchi et al., 1994 . In view of this fatty acid composition and the data reported here, one can deduce that the long chain PUFA requirements of cephalopod paralarvae must be very high. There was a clear influence of the lipid composition of the food on the lipids of cultured octopus paralarvae both at the level of lipid class and fatty acid composition. HB Artemia fed octopuses showed a clear increase in TAG, whereas the octopus paralarvae fed pellets showed a lipid class profile closer to the one of the newly hatched individuals. The paralarvae fed pellets had a higher content of free fatty acids, which were abundant, together with ethyl esters, in the pellets. The influence of the lipid composition of the natural food on the body composition of cephalopods both in the Ž . flesh and in the digestive gland has also been discussed by other authors Joseph, 1989 . The dietary requirements for ny3 PUFA, particularly DHA is critical in early developmental stages due to the high demand for membrane synthesis where the ny3 Ž . PUFA are incorporated Henderson and Sargent, 1985 . DHA plays an important role in Ž maintaining the structural and functional integrity of cell membranes in fish Sargent, . 1995 . This fatty acid may be even more important for the correct development and survival of fast growing phospholipid-rich cephalopod paralarvae. The cultured octopus paralarvae showed a lower content of PUFA as compared with the newly hatched individuals. The differences were particularly marked with DHA and EPA, were probably of dietary aetiology in view of the results obtained when fed pelleted diet, and can be the cause of the poor performance of the paralarval rearing. Fish larvae are strongly dependent on the fatty acids of their prey for the elaboration Ž of major polar lipids in key organs including neural and visual apparatus Navarro et al., . Ž . 1993b, 1995 . Navarro et al. 1993b reported that 30 days of dietary manipulation were sufficient to change the fatty acid composition of the lipids of the heads, eyes and bodies Ž of herring larvae. Similar results were obtained with sea bass larvae Navarro et al., . Ž . Ž 1995 and with weaned turbot Mourente et al., 1991 and sea bream Mourente and . Tocher, 1993 . In view of the data reported here, this dietary effect seems to be a fast Ž . generalised process not limited to fish and crustacean Montano and Navarro, 1996 ˜ Ž . larvae, whose physical poor growth, survival and resistance and physiological signifi- Ž . cance Bell et al., 1995 can perhaps be extrapolated to cephalopod paralarvae. Despite the better lipid class profile and fatty acid composition of group SS, low survival and growth were reached. Since the lower survival of this group was obtained from the beginning of the culture, causes other than the quality of food may be directly involved. In fact, the clogging of the filters at day 8 and a decrease in water quality due to an excess of pellets offered to the paralarvae at day 10 seem to be the causes of peak moralities in cultures HB and SS, respectively. Aside from these considerations, both cultures gave poor growth and survival as compared to other cultures carried out using Ž . crustacean zoeae as food see Villanueva, 1995 , pointing to causes other than simply
  • 14. ( ) J.C. NaÕarro, R. VillanueÕarAquaculture 183 2000 161–177 174 the lipid composition of food for the lack of success in this paralarval culture, perhaps a strong dietary imbalance of the enriched Artemia and difficulties in getting the paralarvae to accept inert food. During the paralarval rearing, it was observed that the paralarvae fed on the pelleted diet. With our experimental design it was impossible to quantify the amount of inert food ingested but it is clear that this had an effect on the lipid composition of paralarvae, which encourages the use of co-feeding techniques to correct the deficient nutrient Ž . composition of Artemia as sole paralarval food Rosenlund et al., 1997 . This is the first time that pellets have been used for rearing paralarval stages of cephalopods. In contrast with larval fishes, cephalopod paralarvae manipulate the food with their arms before ingestion after capture. The high incidence of captured pellets by O. Õulgaris paralarvae but with subsequent low ingestion indicates that palatability could be a determinant factor on the ingestion rates. Although during the present study, low success was obtained with pellet ingestion, co-feeding techniques can be a way in the future for rearing cephalopod paralarvae, provided palatability and feeding protocols are improved. Pelleted diets have been previously used for rearing subadult stages, indicating the Ž adaptability of cephalopods to feed on inert food Lee et al., 1991; Castro et al., 1993; . Castro and Lee, 1994 . 5. Conclusion In view of the present results, Artemia does not seem to be the right choice to feed early stages of cephalopods. From their lipid composition and that of their natural food, it can be deduced that cephalopod paralarvae and juveniles must require a food rich in PUFA, phopholipids and cholesterol and with a moderate content of neutral lipids. This is particularly clear for the squid L. Õulgaris and the cuttlefish S. officinalis. However, these theoretical lipid requirements are common to most fish and crustacean larvae that, on the other hand, are reared with different degrees of success using enriched Artemia nauplii. Fine tuning of enrichment by using enrichment diets high in polar lipids Ž . McEvoy et al., 1996 and PUFA andror co-feeding enriched Artemia with inert diets similar to the pellets used in the present experiments should be the choice to attempt paralarval rearing of cephalopods with minimum guarantees of success. Acknowledgements We appreciate the technical assistance of Mr. J. Riba during the rearing experiences. We are grateful to Miguel A. Montolio and M. Angeles G. Albaladejo who helped in the Ž . lipid analysis. Mr. G. Munoz-Ramos Escola del Mar de Badalona collected the ˜ mysidiacean shrimps. This study was partially funded by a joint research project between the CSIC and the ‘‘Department d’Agricultura, Ramaderia i Pesca’’ of the Catalan Government.
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